A cluster of overlapping quaternary neutralizing epitopes (QNEs) is present on the HIV-1 virus protein envelope spikes. They are composed of portions of the V2 and V3 variable regions of gpl 20 and appear to be stabilized by the intermolecular interactions in the gpl 20 trimer. The goal of Project 2 of this HIVRAD is to create immunogens that can elicit potent neutralizing anti-QNE Abs. Anti-QNE monoclonal antibodies (mAbs) are extremely potent, being 1,000-fold more potent than previously described HIV mAbs. Our team is in a unique position to pursue and achieve this goal since: (i) we have generated and characterized >100 human anti-HIV mAbs, (ii) we described the first human QNE mAb, 2909, (iii) we have successfully developed and used a structure-based rational immunogen design strategy for the production of a panel of V3-scaffold immunogens, and (iv) we have shown these rationally designed immunogens to be capable of inducing cross-clade neutralizing Abs in animals. It is a natural and logical extension of our work to apply our rational design strategy to the development of QNE-scaffold immunogens. Having already determined the crystal structures of two QNE mAbs, human mAb 2909 and rhesus mAb 2.5B, and having created a 3D structural model of the QNE in the context of gpl 20, we now propose to design, engineer, synthesize and test the immunogenicity of QNE-scaffold proteins. We will, in Aim 2.1. refine our model of the QNE by investigating the antigen-antibody interactions of QNE mAbs using biophysical and biochemical methods. In Aim 2.2. we will design and create QNE-scaffold proteins by identifying suitable protein scaffolds and grafting the components of QNE into them. After testing for antigenicity with appropriate mAbs and HIV+ sera, we will, in Aim 2.3, use the recombinant QNE-scaffold proteins to select and characterize new QNE mAbs. In Aim 2.4, we will test the recombinant QNE antigens for immunogenicity in non-human primates to determine their ability to induce potent Abs with protective anti-viral functions. These studies will potentially provide an invaluable reagent for inclusion in an HIV/AIDS vaccine.